Anti-Trypanosomatidae Activity of Essential Oils and Their Main Components from Selected Medicinal Plants

Kinetoplastida is a group of flagellated protozoa characterized by the presence of a kinetoplast, a structure which is part of a large mitochondria and contains DNA. Parasites of this group include genera such as Leishmania, that cause disease in humans and animals, and Phytomonas, that are capable of infecting plants. Due to the lack of treatments, the low efficacy, or the high toxicity of the employed therapeutic agents there is a need to seek potential alternative treatments. In the present work, the antiparasitic activity on Leishmania infantum and Phytomonas davidi of 23 essential oils (EOs) from plants of the Lamiaceae and Asteraceae families, extracted by hydrodistillation (HD) at laboratory scale and steam distillation (SD) in a pilot plant, were evaluated. The chemical compositions of the EOs were determined by gas chromatography-mass spectrometry. Additionally, the cytotoxic activity on mammalian cells of the major components from the most active EOs was evaluated, and their anti-Phytomonas and anti-Leishmania effects analyzed. L. infantum was more sensitive to the EOs than P. davidi. The EOs with the best anti-kinetoplastid activity were S. montana, T. vulgaris, M. suaveolens, and L. luisieri. Steam distillation increased the linalyl acetate, β-caryophyllene, and trans-α-necrodyl acetate contents of the EOs, and decreased the amount of borneol and 1,8 cineol. The major active components of the EOs were tested, with thymol being the strongest anti-Phytomonas compound followed by carvacrol. Our study identified potential treatments against kinetoplastids.


Introduction
Kinetoplastida is a group of flagellated protozoa characterized by the presence of a particular structure, known as a kinetoplast, which is located in the single mitochondrion of the cell and is composed of mitochondrial DNA. Despite kinetoplastids having similarities in their genomic organization and cellular structures, they comprise a large variety of organisms, many of them pathogenic, and transmitted by different vectors, causing different diseases in multiple hosts [1]. Parasites of the genera Leishmania [2] and Trypanosoma [3] cause disease in humans, while pathogens of the genus Phytomonas are capable of infecting plants [4].

Plants and Essential Oil Extraction
The EOs studied in this work were obtained from four species of the Asteraceae family and seventeen species of the Lamiaceae family. The plant species Tanacetum vulgare, Lavandula x intermedia "Grosso", Salvia blancoana, and Thymus mastichina were cultivated in distinct locations of Spain (see Table S1 for locations and voucher numbers). The plant species were identified by Dr. Daniel Gomez, IPE-CSIC, and the seeds were deposited at the CITA (Centro de Investigación y Tecnología Agroalimentaria de Aragón, Unidad de Recursos Forestales, Zaragoza, Spain) germplasm bank. Location, identification, and voucher numbers of the remaining species have been previously reported by Bailén et al. [24].
The EOs were obtained at the Research Center and Food Technology (CITA-Aragón, Spain) using two different methods, hydrodistillation (HD) and steam distillation (SD), as described by Bailén et al. [24]. Aerial plant parts were collected at the flowering stage between 2016 and 2019. The hydrodistillation was carried out in triplicate with 100 g of dried aerial plant parts and 1 L of water for 1 h in a Clevenger-type. The oils were dried over MgSO 4 , filtered and stored at 4 • C until used. Pilot plant steam distillation was carried out on the fresh biomass of the plants (60 kg total fresh plant biomass) harvested at the flowering stage. A stainless-steel pilot extraction plant equipped with a pressure reducing valve was used as described. The pressure of the work was 0.5 bar. The hydrolate (aqueous phase) was decanted from the essential oil collected in the condensation section, and filtered.

EO Analysis
The EOs were analyzed by gas chromatography-mass spectrometry (GC-MS) using a Shimadzu GC-2010 Plus coupled to a Shimadzu GCMS-QP2010-Ultra mass detector with an electron impact ionization source at 70 eV and a Single Quadrupole analyzer, and employing Helium as carrier gas. The samples were injected by an automatic injector (AOC-20i). Chromatography was carried out with a Teknokroma TRB-5 (95%) Dimethyl-(5%) diphenylpolysiloxane capillary column, 30 × 0.25 mm ID and 0.25 µm phase thickness. The working conditions used were: Split mode injection using 1 µL of sample with a split ratio (20:1) employing a Shimadzu AOC-20i automatic injector, injector temperature 300 • C, transfer line temperature connected to the mass spectrometer 250 • C, and ionization source temperature 220 • C. The initial column temperature was 70 • C, heating up to 290 • C at 6 • C/min, and leaving at 290 • C for 15 min. All the samples (4 g/µL) were previously dissolved in 100% dichloromethane (DCM) for injection.
The mass spectra and retention time were used to identify the compounds by comparison with those found in the Wiley database (Wiley 275 Mass Spectra Database, 2001) and NIST 17 (NIST/EPA/NIH Mass Spectral Library), while the relative area percentages of all peaks obtained in the chromatograms were used for quantification. Identification of necrodanes in L. luisieri was performed with our own database (CSIC), built by the injection of pure compounds isolated from this plant [25]. In addition, to confirm the identities of the constituents, the retention index of marker constituents of known EOs were used.

Pure Compounds
After analyzing the composition of the EOs by GC-MS, thymol, carvacrol, γ-terpinene and p-cymene, major components of EOs with anti-Phytomonas activity, were selected for further studies. Some of the major compounds that were excluded were not identified, not commercially available, or not easy to obtain or isolate. Pure compounds (monoterpenes) were obtained from commercial sources. Thymol (≥98.5%) was obtained from Sigma Aldrich (Madrid, Spain), γ-terpinene (97%) and p-cymene (99%) from Acros Organics (Madrid, Spain), and carvacrol (98%) from Fluka (Madrid, Spain).

Anti-Parasitic Activity In Vitro
Anti-Phytomonas (AP) and anti-Leishmania (AL) activity studies were performed on promastigote forms of P. davidi ATCC ® 30287™, isolated from Euphorbia heterophylla [26] and L. infantum "JPC" (MCAN/ES/98/LLM-722), kindly donated by Dr. J.M Requena from CBM-CSIC. The P. davidi promastigotes were cultured in LIT medium and the L. infantum JPC in RPMI medium, supplemented with 10% and 15% heat-inactivated fetal calf serum (FCS), respectively, at 28 • C. RPMI was also supplemented with 10 µg/mL of hemin (Acros Organics, Madrid, Spain). Parasites in the logarithmic growth phase were distributed in 96-well flat-bottom plates (95 µL of culture/well). EOs and compounds in DMSO were tested in quadruplicate at various concentrations for 24 h (Phytomonas) and 48 h (Leishmania) at several concentrations (EOs at 800, 400 and 200, 100, 50 and 25 µg/mL; and pure compounds at 100, 50, 25, 10 and 1 µg/mL). Amphotericin B was used as a reference drug. The parasite viability was analyzed by a modified MTT colorimetric assay method [27]. The percentage of AP activity and AL activity was calculated as growth inhibition using the following formula: % AP or AL = 100 − [(Ap − Ab) ÷ (Ac − Ab)] × 100; Ap is the absorbance of the tested product, Ab the absorbance of the blank, and Ac the absorbance of the control wells (not treated).

Cytotoxicity of Pure Compounds
African green monkey kidney cells (Vero cells) were maintained in Dulbecco's modified Eagle's minimal essential medium (DMEM) supplemented with 10% fetal calf serum and 1% penicillin/streptomycin (Fisher Scientific, Madrid, Spain) at 37 • C under a humidified atmosphere of 5% CO 2 /95% air.
Cells seeded in 96-well flat-bottom microplates with 100 µL medium per well (initial densities 10 4 cells per well) were exposed for 48 h to serial dilutions (100, 75, 50, 25, 10 and 1 µg/mL) of the tested compounds in DMSO (< 1% final concentration). The cell viability was analyzed by the MTT colorimetric assay method, and the purple-colored formazan precipitate was dissolved with 100 µL of DMSO [24].

Statistical Analysis
The data were analyzed using STATGRAPHICS Centurion XIX (https://www.statgraphics. com, accessed on 5 September 2022). A parametric bivariate correlation analysis was performed between the main components of the EOs, present in a proportion higher than 5%, and the AL or AP activity (IC 50 ).
The cells' and promastigotes' viabilities were tested with each compound in a doseresponse experiment, to calculate their relative potency (CC 50 or IC 50 value). IC 50 (µg/mL) expresses the dose of EOs or pure compounds needed to produce 50% mortality of promastigotes, while CC 50 (µg/mL) expresses the dose of compounds necessary to produce 50% mortality of Vero cells.
The selectivity index was calculated for the AP or AL of pure compounds, using the formula SI = IC 50 /CC 50 . Compounds with an SI higher than one were considered as potential anti-Phytomonas or anti-Leishmania compounds, since they are more toxic for kinetoplastids than for mammalian cells.

Antiprotozoal Activity of EOs from Lamiaceae and Asteraceae Plants
The activity of 23 EOs from 17 selected species (domesticated or undergoing domestication) belonging to the Asteraceae and Lamiaceae families were evaluated against the kinetoplastids P. davidi and L. infantum. All of these EOs were obtained at laboratory scale by hydrodistillation (HD). EOs from the domesticated plants cultivated in the field (a total of 17) were also extracted at a semi-industrial scale, representative of a potential commercial situation, by steam distillation (SD) ( Table 1). Eight EOs, from five Lavandula species, were tested against Leishmania. Six of them showed high antileishmanial activities at 800 µg/mL (AL > 70%), one had a moderate effect (AP: 50-70%), and one was not active ( Figure 1A and Figure S1A). The most effective Lavandula EOs were L. luisieri 1 (SD) and L. luisieri 2 (HD and SD), followed by L. mallete (SD) and L. intermedia "Abrial" (SD). The EOs with the highest anti-Phytomonas activity were L. intermedia "Super", L. luisieri 1 (HD), and L. mallete (HD). Overall, EOs obtained by SD from Lavandula sp. were more active against Leishmania and those extracted by HD were more active against Phytomonas.
Lavandula sp. were more active against Leishmania and those extracted by HD were more active against Phytomonas. EOs belonging to S. montana, M. suaveolens, and R. officinalis had effects on both parasites. Differences related to the EO extraction method were observed for R. officinalis against both parasites (Figures 1 and S1). S. montana EOs had the highest antiprotozoal activity against both parasites and the AP activity was maintained even at 100 µ g/mL.  EOs belonging to S. montana, M. suaveolens, and R. officinalis had effects on both parasites. Differences related to the EO extraction method were observed for R. officinalis against both parasites (Figure 1 and Figure S1). S. montana EOs had the highest antiprotozoal activity against both parasites and the AP activity was maintained even at 100 µg/mL. Among the four EOs from Salvia spp., two were highly active against L. infantum, and two had no activity ( Table 1). The EOs with the largest effects were S. hybrid (HD) and S. officinalis (SD). Worse results were obtained against Phytomonas, with three EOs having moderate effects and one showing no activity. S. officinalis was the Salvia species with the highest anti-Trypanosomatidae activity. The EOs from Origanum and Thymus spp. showed high or moderate activity against Leishmania. The best AL effects were obtained for T. zygis, and T. vulgaris (SD EOs). On P. davidi, T. vulgaris (HD) showed the highest activity at 200 µg/mL. No activity against Phytomonas was found for T. mastichina, T. zygis (HD), or O. majorana.
The EOs from the Asteraceae (Tanacetum, Santolina, and Ditrichia) showed moderate antiprotozoal activities, except for T. vulgare which showed no activity on P. davidi and L. infantum.

Chemical Composition of Essential Oils
EOs with an IC 50 lower than 200 µg/mL were selected for further analysis (both extraction methods), adding to a total of six species and seven EOs ( Table 2 and Table S2; Figures S2-S14). Only components with a proportion higher than 5% were considered. Table 2. Main components of the EOs from the most active species (abundance ≥ 5%).
Camphor, trans-α-necrodyl acetate, and fenchone were predominant in L. Luisieri (1 and 2), with fenchone being more effectively extracted by HD and trans-α-necrodyl acetate by SD. For M. suaveolens, the main components were piperitenone and piperitenone oxide, which accounted for more than 60% of the total composition. Piperitenone was more abundant in the EO obtained by HD and piperitenone oxide in the EO obtained by SD. Among the components of S. hybrid HD, 1,8 cineol, camphor, and trans-bornyl acetate appeared in higher proportions.
In the chemical composition of S. montana, thymol, p-cymene, and carvacrol stand out, with thymol and p-cymene being more abundant when extracted by HD and carvacrol when the extraction was done by SD. Thymol and p-cymene were also the main components of T. vulgaris and T. zygis. Linalyl acetate was also an abundant component, but only for T. zygis SD EO.
A parametric bivariate correlation analysis was performed between the main components of the EOs and the AL and AP activities. There were two significant correlations for AL and three for AP (p < 0.05) ( Table 3). Among the compounds with significant correlations, piperitenone oxide was directly associated with the AL activity (negative correlation between compound abundance and IC 50 of the EO), whereas p-cymene, thymol, and carvacrol were directly associated with the AP activity of the EOs. Only α-terpineol was inversely associated with AL activity (positive correlation between compound abundance and IC 50 of the EO).

Anti-Phytomonas and Cytotoxic Activity of Pure Compounds
A total of four compounds (Figure 2), selected from the chemical composition of the most active EOs, were tested on L. infantum and P. davidi, and also their cytotoxic effects on Vero cells were evaluated (Table 4). Amphotericin B was used as a reference drug. Data previously reported from these compounds on Leishmania sp. have been included in Table 4. Only compounds 1 (thymol) and 2 (carvacrol) showed AP activities with moderate effects, with 1 (thymol) being the compound with the highest AP activity (IC 50 : 45 µg/mL). The highest SI was observed for thymol (SI: 2.22). Compounds 1 (thymol) and 2 (carvacrol) were also the terpenes with the highest selectivity indexes (SI: 10.20 and 13.85 respectively) on L. infantum. p-Cymene (4) was the only terpene previously tested on other Leishmania species (L. chagasi) [28]. None of the tested compounds were cytotoxic against Vero cells.

Anti-Phytomonas and Cytotoxic Activity of Pure Compounds
A total of four compounds (Figure 2), selected from the chemical composition of the most active EOs, were tested on L. infantum and P. davidi, and also their cytotoxic effects on Vero cells were evaluated (Table 4). Amphotericin B was used as a reference drug. Data previously reported from these compounds on Leishmania sp. have been included in Table  4. Only compounds 1 (thymol) and 2 (carvacrol) showed AP activities with moderate effects, with 1 (thymol) being the compound with the highest AP activity (IC50: 45 µ g/mL). The highest SI was observed for thymol (SI: 2.22). Compounds 1 (thymol) and 2 (carvacrol) were also the terpenes with the highest selectivity indexes (SI: 10.20 and 13.85 respectively) on L. infantum. p-Cymene (4) was the only terpene previously tested on other Leishmania species (L. chagasi) [28]. None of the tested compounds were cytotoxic against Vero cells.  Vero Cells [25] P. davidi L. infantum Figure 2. Chemical structures of the tested monoterpenes. 10.000 a CC50 (µg/mL) = concentration needed to produce 50% Vero cell mortality; b IC50 (µg/mL) = concentration needed to produce 50% trophozoite mortality, c SI: Selectivity index.; nd: not determined.

Discussion
Lamiaceae is an important family, with a variety of aromatic and medicinal genera such as Rosmarinus, Origanum, Thymus, Lavandula, Mentha, and Satureja, among many others [31]. The most active EOs found in our study for both kinetoplastids, L. infantum and P. davidi, belong to this family, highlighting EOs from M. suaveolens and S. montana. The Asteraceae plant family has emerged as a new source of trypanocidal and leishmanicidal compounds [32]. However, the Asteraceae EOs tested in this study only had moderate effects on both targets.
Overall, the results of the tested EOs in our study showed that L. infantum was more sensitive to the EOs than P. davidi, probably because Phytomonas is a plant parasite, and thus it is more adapted to the chemical defensive components of plants. These results agree with those found by Sainz et al. when comparing the activity of various EOs on T. cruzi and P. davidi [27]. The activity of EOs on Phytomonas sp. have been less studied than the leishmanicidal effects. Even so, EOs from other plant families have been found to have anti-Phytomonas activity. EOs from Varronia curassavica genotypes have antiprotozoal activity against Phytomonas serpens causing alterations in the permeabilization of the cytoplasmic membrane [33]. Lantana camara EOs, extracted at different harvesting times, displayed trypanocidal activity on P. serpens [34], and EOs from Hyssopus officinalis showed antiprotozoal activity on T. cruzi and P. davidi [35]. However, this is the first report on the phytomonacidal effects of the EOs studied here.
M. suaveolens is an aromatic species usually employed in traditional medicine, with reported cytotoxic, antioxidant, anti-inflammatory, antifungal, antiviral, and insecticidal properties [36]. Our results indicate that M. suaveolens EO had the highest antileishmanial effects of all the tested EOs, as well as moderate effects on Phytomonas. This EO contained high proportions of piperitenone oxide and piperitenone. Piperitenone oxide could be involved in the antileishmanial activity, as it has been previously been reported to have trypanocidal, insecticidal, and schistosomicidal effects [37][38][39]. There are also other species of the genus Mentha with antileishmanial properties on promastigotes of various species of Leishmania. EOs from M. australis and M. microphylla have antileishmanial effects on L. major [40], M. pulegium on L. major, L. infantum and L. tropica [41], M. x piperita on L. infantum and L. donovani [42,43], and M. cervina on L. infantum [42,44]. However, none of the mentioned EOs had piperitenone oxide as a major component.
EOs from two populations of L. luisieri (1 and 2) had the strongest effects among the tested lavandulas on L. infantum. Essential oils from plants of the Lavandula genus have reported acaricidal, antibacterial, antifungal, antioxidant, and anti-parasitic effects [53]. EOs from L. angustifolia, L. stoechas, L. viridis, and L. luisieri had reported effects on L. major, L. infantum, and L. tropica [42,[53][54][55]. However, the effect found here was weaker than that observed by Machado et al. on L. infantum promastigotes [53]. Trans-α-necrodyl acetate is one of the major components of L. luisieri EOs. Therefore, necrodane derivatives should be further studied to verify their antileishmanial properties. L. luisieri EOs can produce leishmanicidal effects through different mechanisms, but mainly through unleashed apoptosis, with phosphatidylserine externalization, mitochondrial membrane potential loss, and G0/G1 phase cell cycle arrest being the most remarkable aspects involved in apoptosis [53]. On P. davidi, the studied Lavandula EOs only had moderate effects, with L. intermedia "Super" being the most active.
T. vulgaris HD EO, showing a remarkable anti-Phytomonas activity, had the highest content of thymol (48%) of all the tested EOs. As mentioned before, thymol correlated with AP activity. EOs from T. zygis, Thymus capitelatus, and T. mastichina have been reported to have antileishmanial effects on L. infantum promastigotes [42,56].
Among the genus Salvia, the EOs from S. hybrid and S. officinalis showed important antileishmanial effects, while S. blancoana EOs had moderate action against P. davidi. EOs from various species of Salvia have been previously tested on Phytomonas and Leishmania, with moderate effects [17,22].
In this study, the EO (HD and SD) from R. officinalis showed a moderate effect on L. infantum. Previous reports have showed strong antileishmanial effects of R. officinalis EOs on L. major, L. tropica, and L infantum [41,54]. Variations in the chemical composition of the EOs could explain these differences.
Origanum majorana is a shrub found in Asia and in the Mediterranean area, with reported antibacterial, antifungal, antiparasitic, anthelmintic, and antiviral activities [57][58][59]. EOs from O. majorana and O. dubium have been reported to have antimalarial effects on mice, reducing the parasitemia and increasing their life span [57]. Also, EOs from O. majorana, O. virens, and O. vulgare have been reported to have anti-Phytomonas effects on P. davidi, with O. virens having the strongest effect [22]. Other antileishmanial effects were reported from O. virens EO on L. infantum [42], and O. vulgare EO on L. amazonensis, L. panamensis, and L. braziliensis [60,61].
In our study, thymol (1) and carvacrol (2) correlated with AP activity, and were active when tested on P. davidi, while p-cymene was not. Thymol and carvacrol are p-cymene derivatives, and therefore their amounts in EOs are usually correlated, explaining the correlation of this compound with AP effects. Compounds 1 and 2 are common in EOs with activity against promastigotes and amastigotes of L. infantum [29]. Thymol has been reported to have anti-Phytomonas [22] and leishmanicidal properties on promastigotes of L. infantum [29], promastigotes and amastigotes of L. infantum, and amastigotes of L. donovani [62]. The anti-Leishmania and anti-Phytomonas activities of carvacrol and thymol depend on the presence of the phenolic hydroxyl group, as observed before by Silva et al. [63]. The lack of the phenolic hydroxyl group in the precursor p-cymene is associated with the absence of antiprotozoal effects. Also, thymol has been used as a starting compound to obtain derivatives with stronger antileishmanial effects [64].
The biological properties of EOs can be determined by their major compounds, but minor compounds modulate these effects. Synergistic effects between EO components have been observed before. A mixture 1:4 of lupenone and β-caryophyllene oxide presented better antileishmanial activity and lower cytotoxicity than β-caryophyllene oxide alone [65]. Also, the combination between ascaridole and carvacrol produced synergistic effects on L. amazonensis [66].

Conclusions
EOs with better anti-kinetoplastid activity were S. montana, T. vulgaris, M. suaveolens, and L. luisieri. They are good sources of thymol, carvacrol, trans-α-necrodyl acetate, and piperitenone oxide. Further studies should be performed with trans-α-necrodyl acetate and piperitenone oxide to corroborate their potential activity against L. infantum and P. davidi. Thymol and carvacrol were the best anti-kinetoplastid compounds of this study. The synthesis of new compounds, using carvacrol and thymol as starting compounds, could be a strategy for the search for new anti-trypanosomatid compounds as alternatives to the current treatments, due to their anti-Phytomonas and antileishmanial effects.
Phytomonas is a plant parasite that can cause important economic losses and that lacks an effective treatment. Our study identifies potential treatments against this pathogen, and extraction methods which potentiate the concentration of specific components. The potential use of EOs, and their main components, as new alternatives for the treatment of animal trypanosomatid diseases, including leishmaniasis, could lie in their use as alternative treatments or in combination with approved treatments, to increase the efficiency and diminish the toxic effects of the current therapeutic protocols.